ITRM940625A1 - USE OF CARNITINE AND ALCANOYL CARNITINE PHTALIDYLIDEN ESTERS FOR ENDOTOXIC SHOCK TREATMENT - Google Patents

Description

Description of the industrial invention entitled:

“Use of phthalidylidene esters of carnitine and alkanoyl carnitine for the treatment of endotoxic shock”.

The present invention relates to the use of alkyl (3-phthalidylidene) esters of carnitine and alkanoyl carnitine to prepare pharmaceutical compositions suitable for the treatment of endotoxic shock. These esters have general formula (I):

in which:

Y is an alkylene radical having 1-5 carbon atoms, unsubstituted, or substituted with one or more C1 -C4 lower alkyl groups;

R is hydrogen or alkanoyl having from 2 to 6 carbon atoms;

R1, R2, R3 and R4, the same or different, are hydrogen, halogen, alkoxy with 1-8 carbon atoms, alkyl less than 1-4 carbon atoms, alkyl

lower halogen-substituted, amino or amino alkyl-substituted wherein the alkyl radical has 1-4 carbon atoms, nitro, cyano, acylamino having 1-4 carbon atoms, or Ri and R2 together, R2 and R3 together 0 R3 and R4 together form an alkylenedioxy group having 1-4 carbon atoms; And

X is the anion of a pharmacologically acceptable acid.

In particular, the various substituents can assume the following meanings in formula (I):

Y is alkylene with 1-2 carbon atoms:

R is selected from hydrogen, acetyl, propionyl, butyryl, isobutyryl, valeryl and isovaleryl;

R1, R2, R3 and R4, the same or different, are selected from hydrogen, fluorine, chlorine, methoxy, ethoxy, methyl, trifluoromethyl, trichloromethyl, amino, nitro, cyano and acetylamino.

Χ is the anion of a pharmacologically acceptable acid selected from chloride:

bromide; iodide; aspartate, particularly acid aspartate; citrate, particularly acid citrate; phosphate, particularly acid phosphate; fumarate, particularly acid fumarate; glycerophosphate; glucose phosphate; lactate; maleate, particularly acid maleate; orotate; oxalate, particularly acid oxalate; sulfate, particularly acid sulfate; trichloroacetate; trifluoroacetate and methanesulfonate.

According to the present invention, the following phthalidylidene derivatives are particularly preferred:

(Z) - [3- (5-chlorophthalidylidene) ethyl ester] of isovaleryl L-carnitine bromide (ST 899);

(Z) - (3-phthalidylidene) ethyl ester of isovaleryl L-carnitine bromide

(ST 776);

(Z) - [3- (6-methoxyphthalidylidene) ethyl] ester of isovaleryl L-carnitine bromide (ST 867);

(Z) - [3- (6-fluorophthalidylidene) ethyl ester] of isovaleryl L-carnitine bromide (ST 900);

(Z) - [3- (6-iluoroophthalidylidene) ethyl] ester of propionyl L-carnitine bromide (ST 926);

(Z) - [3- (7-chlorophthalidylidene) ethyl] ester of isovaleryl L-carnitine bromide (ST 954); And

(Z) - [3- (6-methylphthalidylidene) ethyl ester] of isovaleryl L-carnitine bromide (ST 1063).

The esters of general formula (I) are known compounds; see eg. EP 0 488 965 A1 where they have been described as potent antagonists of PAF (platelet-activating factor) and acetylcholinesterase and therefore useful as active ingredients in pharmaceutical compositions for the treatment of pathological states of which PAF is the agent or one of the etiological agents. Furthermore, in view of their antagonism towards acetylcholinesterase, they have been described as active in the treatment of pre-senile and senile dementias (typically, Alzheimer's disease). The same EP 0 488 965 A1, which is incorporated by reference in the present description, describes the physicochemical characteristics and the procedures for the preparation of the compounds (I).

It has now been found that the esters of formula (I) are active in the treatment of endotoxic shock. This activity could not be predicted on the basis of the activities already known for these compounds.

Endotoxic shock is a clinical syndrome associated with high mortality, and characterized by various hemodynamic, immunological and biochemical alterations.

Its growing incidence places it among the most serious hospital pathologies, especially in intensive care units despite the use of various antibiotics, surgical drainage, surgery with vasoactive substances and metabolic support. It is estimated that about 100,000 people die annually in the US from endotoxic shock.

The main cause of this type of pathology is certainly the serious infection by Grani 'bacteria, whose pathophysiological effects are attributable to the LPS, a component of the outer layer of the bacterial membrane capable of causing endotoxic shock by interacting with various components of the immune system. host, especially with macrophages.

This immunocompetent cell population in fact releases different endogenous mediators which are ultimately responsible for the complex pathological picture that follows.

The fatal outcome of endotoxic shock in humans has recently been associated with the systemic release of large quantities of various cytokines. In fact, there are numerous studies that show that an abnormal modulation of cytokines such as IL-1, IL-6, TNF and IFNy is closely connected with a serious situation of sepsis.

In any case, TNF (Tumor Necrosis Factor) is the cytokine that plays a crucial role as a mediator in the host response to LPS (Tracy K. J. Tumor Necrosis Factor (Cachectin) in the Biology of Septic Shock Syndrome. Circshòck 1991: 35: 123-128), having been shown to be involved in various metabolic alterations that characterize the course of shock (Starnes H. F., Warren R. S. Jeevanandam M. et al., Tumor Necrosis Factor and the acute metabolic response to tissue injury in man. J. Clin. Invest. 1988: 82: 1321), a poor prognosis of which is often related to excessively high serum TNF concentrations (Dames P., Reuter A., Gysen P., Demonty J., Lamy M., Franchimont P. Tumor Necrosis Factor and interleukin- 1 serum levels during severe sepsis in humans, Crit. Care Med. 1989: 17: 975-978. Debets J. M. H., Kampmeijer R. Van der Linden M. P. M. H., Buurman W. A. Van der Linden C. J. Plasma Tumor Necrosis Factor and mortality in critically ili septic p careful. Crit. Care Med., 1989: 17: 489-494).

In fact, high levels of TNF are found in the serum of animals experimentally intoxicated with LPS, and animals directly inoculated with TNF undergo a toxic syndrome indistinguishable from endotoxin (Natanson C., Eichenhols P. W., Danner R. L., Endotoxin and Tumor Necrosis Factor challenges in dogs simulate the cardiovascular profile of human septic shock. J. Exp. Med. 1989: 169: 823-832; Beutler B., Milsark I. W., Cerami A., Passive immunization against cachectin / Tumor Necrosis Factor protects mice from lethal effects of endotoxin , Science 1985: 229: 869-871).

Consequently, compounds that block or antagonize TNF may be considered useful therapeutic candidates in the treatment of endotoxic shock.

It has been found that the esters of formula (I) are endowed with such pharmacological properties.

EVALUATION OF THE EFFECT OF THE (Z) - [3- (5-Chlorophthalidyliden) -ethyl ester] of isovaleryl L-carnitine bromide (ST 899) ON THE SILK LEVELS OF TNF AND ON THE LPS-INDUCED MORTALITY IN C57BL / 6 MICE.

Inbred male C57BL / 6 mice (Iffa Credo) aged 7 weeks (8-10 animals per experimental group) were used.

The compound ST 899, solubilized at 37 ° C in sterile physiological solution, was administered according to 2 different treatment protocols with respect to the i.p. of LPS (30 mg / kg at time 0), namely:

Protocol A: at -60 'per os and at -10' i.v.

Protocol B: at -60 'i.p. and at -10 'i.v.

The doses used ranged from 1.25 to 5 mg / kg and were chosen based on acute toxicity studies in mice.

In our experimental conditions, the animals treated i.v. with the higher dose (5 mg / kg), they experienced loss of reflexes and occasional seizures from which they recovered within 5 minutes of i.v. administration. No alteration was observed when the animals were treated i.v. with lower doses, i.e. 2.5 and 1.25 mg / kg, nor when the 5 mg / kg dose was administered orally or i.p ..

The LPS used is E. coli 055: B5 lipopolysaccharide and has been dissolved in physiological solution.

Essay of the TNF

Blood samples were all taken 1 hour after LPS administration, time in which maximal levels of circulating TNF are reached. Blood was collected from the retro-orbital plexus of previously anesthetized animals by short CO2 inhalation Blood samples were incubated 2 hours at T. A. and the resulting serum was then centrifuged at 4000 rpm for 20 minutes before being frozen at - 80 ° C pending the dosing of the serum TNF.

TNF activity was tested using L929 tumor cells (a murine fibrosarcoma), which were found to be particularly sensitive to the cytotoxic action of TNF.

In detail, L929 cells were seeded at a density of 3.2x10 cells / mL into the wells of a flat bottom microtiter plate (0.1 mL / well) using RPMI-1640 culture medium containing 10% FCS. After 18-24 hours of incubation at 37 ° C in a humidified thermostat with 5% CO2, the culture supernatant was removed and 0.1 ml of scaled dilutions, carried out in RPMI containing 1% FCS, of the samples were added to the wells in triplicate. silky.

0.1 ml of a solution containing 2 μg / ml of Actinomycin D was then added to all wells. The function of this cellular RNA biosynthesis inhibitor is to amplify the sensitivity of L929 cells to the cytotoxic effect of TNF.

After further 18 hours of incubation at 37 "C in a humidified thermostat with 5% C02, the supernatants were aspirated and the cell monolayers were washed 3 times with physiological solution. The cells adhered to the bottom of the wells were stained for 15 minutes. with a solution of Cristal violet (0.25% Cristal violet, 20% ethanol, 8% formalin and 100% H20 q.s.).

After staining was complete, the dye was aspirated and the cells were gently washed with tap water. After allowing the microtiter wells to dry completely, the dye taken from the cells was eluted with a 33% acetic acid solution and the absorbance of the samples was determined by a microtiter plate spectrophotometer (Multiskan MCC 340) using the filter at 570 nm.

The values obtained were then used to determine the TNF units present in each serum sample by direct comparison of the regressions obtained with the serial dilutions of the various samples with the regression obtained using various dilutions of a recombinant murine TNF standard at known concentration.

Statistic analysis

The evaluation of the significance of the results relative to the levels of TNF was carried out with the Student "t" test. Survival data were analyzed with Fisher's exact test.

RESULTS

Effect of ST 899 on LPS-induced release of serum TNF.

The results obtained with the administration of 5 mg / kg of ST 899 according to the experimental protocols A and B are reported in Table 1. It can be noted that the ST 899 significantly decreases the level of circulating TNF induced by the LPS.

A subsequent experiment carried out by administering ST 899 according to protocol B revealed that a reduction in the serum level of TNF of the same order of magnitude can also be obtained using lower doses of ST 899.

In fact, in addition to the significant reduction (p <0.00-l) obtained with 5 mg / kg, at doses of 1.25 and 2.5 mg / kg there was a significant decrease (p <0.01) in circulating TNF equal to 76% and 86 respectively. % of LPS control (Tab. 2).

Effect of ST 899 on LPS-induced mortality.

The results of a first experiment aimed at examining the effects of treatment with ST 899 on LPS-induced mortality are reported in Table 3.

In this case the treatment with ST 899 (5 mg / kg) was carried out according to Prot. B, that is to say by administering the substances at -60 minutes (i.p.) and at -10 minutes (i.v.) compared to the challenge with LPS (30 mg / kg).

It can be seen that, with these dosages and this administration scheme, ST 899 is able to protect mice from LPS-induced mortality. In fact, the survival of animals treated with ST 899 significantly increased (p <0.02) reaching 66% compared to 11% in the LPS control.

Likewise, the MST is significantly prolonged (Tab. 3 and Fig. 1) by the ST 899 (p <0.002) reaching a median value equal to l l for both substances, considerably higher than that of the LPS (MST = 3 days).

Some protection, in terms of both survival and STD, is also found in animals treated with lower doses of ST 899, i.e. 1.25 and 2.5 mg / kg, although not statistically significant (Table 4 and Fig. 2 ).

It can also be noted that, confirming the initial experiment, treatment with 5 mg / kg of ST 899 is again protective, significantly increasing both survival and MST (pcO.OOl and p <0.02, respectively).

The experiments carried out indicate that the substance ST 899 is endowed, even at a dose as low as 1.25 mg / kg, with a strong inhibitory activity on the production of TNF induced by LPS.

To achieve significant effects on animal survival, however, a higher dose (5 mg / kg) must be administered. This result indicates that in our experimental model, in addition to TNF, other mediators are involved as effectors of the cascade of events leading to endotoxin-induced lethality.

Table 1 Effect of ST 899 administration on serum TNF levels (U / ml) induced by inoculation with LPS (30 mg / kg, i.p.).

Table 2 Effect of ST 899a administration on serum TNF levels (U / ml) induced by inoculation with LPS (30 mg / kg, i.p.).

'

Figure 1 Effect of treatment with ST 899 on lethality induced by i.p. of 30 mg / kg of LPS (day 0). The treatment was carried out by administering to mice (9 animals per group) 5 mg / kg of ST 899 60 minutes before (i.p.) and 10 minutes before (i.v.) the LPS challenge.

Figure 2 Effect of treatment with ST 899 on lethality induced by i.p. of 30 mg / kg of LPS (day 0). Treatment with ST 899 was performed by administering ST 899 to mice (10 animals per group) 60 minutes before (i.p.) and 10 minutes before (i.v.) the LPS challenge.

EVALUATION OF THE EFFECTS OF COMPOUND ST 899 ON LPS-INDUCED MORTALITY IN C57BL / 6 MICE SENSITIZED WITH D-GALACTOSAMINE.

Male inbred C57BL / 6 mice (Iffa Credo) aged 6-8 weeks were used.

The compound ST 899, solubilized at 37 ° C in sterile physiological solution, was administered according to the treatment protocol shown in Table 5.

The lipopolysaccharide (LPS) used is from the bacterium E. coli, namely the serotype 055: B5.

LPS 055: B5 was administered i.p. at a dose of 0.01 mg / kg to mice that had been treated 30 minutes before with the sensitizing agent D-galactosamine (1 g / kg i.v.).

Effect of ST 899 on LPS 055: B5-induced mortality in mice sensitized with D-galactosamine.

Table 5 shows the data relating to the effects of ST 899 on induced mortality in C57BL / 6 mice in the D-galactosamine LPS model (055: B5). In this model, as reported in the literature (Galanos C. and Freudenberg M., Immunobiol, 187: 346-356, 1993), pretreatment with the hepatotoxic agent D-galactosamine considerably amplifies the host's sensitivity to the effects of 'LPS.

In this model, the treatment with ST 899 carried out i.p. 120, 60 and 10 minutes before the LPS challenge was ineffective at the 1.5 mg / kg dose, but was significantly (p <0.05) effective at the 15 mg / kg dose in protecting mice from LPS-induced lethality. in this experimental model.

Table 5 Effect of treatment with ST 899, in the D-galactosamine model LPS 055: B5 in C57BL / 6 mice D-galactosamine (1 g / kg) was administered i.v. 30 minutes before the LPS challenge (0.01 mg / kg, i.p.>. The treatment with 1.5 and 15 mg / kg ST 899 was carried out endoperitoneally 120, 60 and 10 minutes before the LPS challenge.

The compounds of the present invention are administered orally or parenterally, in any of the usual pharmaceutical forms which are prepared by conventional procedures, well known to those skilled in these techniques. These forms include

oral unit dosage forms, both solid and liquid, such as for example tablets, capsules, solutions, syrups and the like, and injectable forms such as for example sterile solutions for ampoules and ampoules.

The usual solvents, diluents and excipients are used for these pharmaceutical forms. Optionally, preservatives, sweeteners and flavoring substances can be added. Non-limiting examples of such substances are sodium carboxymethylcellulose, polysorbate, mannitol, sorbitol, starch, avicel, talc and others which will be evident to those skilled in the pharmaceutical art.

The dose that is administered will be determined by the attending physician taking into account the age, weight and general condition of the patient, based on an appropriate professional evaluation. Although effective results may even be seen at doses of 5 to 8 mg / kg of body weight per day, a dose of between about 10 and about 50 mg / kg of body weight is preferred. Should it be deemed necessary, larger doses may be administered, due to the low toxicity of the compounds of the invention.

Not limitingly, depending on the pharmaceutical form of administration, the dosages provided are as follows:

Claims (9)

CLAIMS 1. Use of alkyl (3-phthalidylidene) esters of carnitine and of alkanoyl carnitine of general formula (I) in which: Y is an alkylene radical having 1-5 carbon atoms, unsubstituted, or substituted with one or more C1-C4 lower alkyl groups; R is hydrogen, or alkanoyl having from 2 to 6 carbon atoms; R1, R2, R3 and R4, equal or different, are hydrogen, halogen, alkoxy with 1-8 carbon atoms, alkyl lower than 1-4 carbon atoms, lower halogen-substituted alkyl, amino 0 amino alkyl-substituted in which the alkyl radical has 1-4 carbon atoms, nitro, cyano, acylamino having 1-4 carbon atoms, or R1 and R2 together, R2 and R3 together or R3 and R4 together form an alkylenedioxy group having 1-4 carbon atoms ; And X 'is the anion of a pharmacologically acceptable acid, to produce pharmaceutical compositions suitable for the treatment of endotoxic shock. Use of esters according to claim 1, wherein Y is methylene or ethylene. 3. Use of esters according to claim 1 or 2, wherein R is selected from acetyl, propionyl, butyryl, isobutyryl, valeryl and isovaleryl. 4. Use according to claim 1, wherein the ester of formula (I) is the (Z) - [3- (5-chlorophthalidylidene) ethyl ester] of isovaleryl L-carnitine bromide (ST 899); 5. Use according to claim 1, wherein the ester of formula (I) is the (Z) - (3-phthalidylidene) ethyl ester of isovaleryl L-carnitine bromide (ST 776); 6. Use according to claim 1, wherein the ester of formula (I) is the (Z) - [3- (6-methoxyphthalidylidene) ethyl ester] of isovaleryl L-carnitine bromide (ST 867); 7. Use according to claim 1, wherein the ester of formula (I) is the (Z) - [3- (6-fluorophthalidylidene) ethyl ester] of isovaleryl L-carnitine bromide (ST 900); 8. Use according to claim 1, wherein the ester of formula (I) is the (Z) - [3- (6-fluorophthalidylidene) ethyl ester] of propionyl L-carnitine bromide (ST 926); 9. Use according to claim 1, wherein the ester of formula (I) is the (Z) - [3- (7-chlorophthalidylidene) ethyl ester] of isovaleryl L-carnitine bromide (ST 954); And Use according to claim 1, wherein the ester of formula (I) is the (Z) - [3- (6-methylphthalidylidene) ethyl ester] of isovaleryl L-carnitine bromide (ST 1063).